Mast Cells Play No Role in the Pathogenesis of

Postoperative Ileus Induced by Intestinal Manipulation

Pedro J. Gomez-Pinilla1, Giovanna Farro1, Martina Di Giovangiulio1, Nathalie Stakenborg1,
Andrea Némethova1, Annick de Vries1, Adrian Liston2,3, Thorsten B. Feyerabend4, Hans-
Reimwer Rodewald4, Guy E. Boeckxstaens1, Gianluca Matteoli1*
1 Department of Clinical and Experimental Medicine, Translational Research Center for Gastrointestinal Disorders (TARGID), KU Leuven, Leuven, Belgium, 2 Department of
Microbiology and Immunology, KU Leuven, Leuven, Belgium, 3 Autoimmune Genetics Laboratory, Vlaams Instituut voor Biotechnologie (VIB), Leuven, Belgium, 4 Division
for Cellular Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany

Abstract
Introduction: Intestinal manipulation (IM) during abdominal surgery results in intestinal inflammation leading to
hypomotility or ileus. Mast cell activation is thought to play a crucial role in the pathophysiology of postoperative ileus
(POI). However, this conclusion was mainly drawn using mast cell-deficient mouse models with abnormal Kit signaling.
These mice also lack interstitial cells of Cajal (ICC) resulting in aberrant gastrointestinal motility even prior to surgery,
compromising their use as model to study POI. To avoid these experimental weaknesses we took advantage of a newly
developed knock-in mouse model, Cpa3Cre/+, devoid of mast cells but with intact Kit signaling.

Design: The role of mast cells in the development of POI and intestinal inflammation was evaluated assessing
gastrointestinal transit and muscularis externa inflammation after IM in two strains of mice lacking mast cells, i.e. KitW-sh/W-sh
and Cpa3Cre/+ mice, and by use of the mast cell stabilizer cromolyn.

Results: KitW-sh/W-sh mice lack ICC networks and already revealed significantly delayed gastrointestinal transit even before
surgery. IM did not further delay intestinal transit, but induced infiltration of myeloperoxidase positive cells, expression of
inflammatory cytokines and recruitment of monocytes and neutrophils into the muscularis externa. On the contrary,
Cpa3Cre/+ mice have a normal network of ICC and normal gastrointestinal. Surprisingly, IM in Cpa3Cre/+ mice caused delay in
gut motility and intestinal inflammation as in wild type littermates mice (Cpa3+/+). Furthermore, treatment with the mast cell
inhibitor cromolyn resulted in an inhibition of mast cells without preventing POI.

Conclusions: Here, we confirm that IM induced mast cell degranulation. However, our data demonstrate that mast cells are
not required for the pathogenesis of POI in mice. Although there might be species differences between mouse and human,
our results argue against mast cell inhibitors as a therapeutic approach to shorten POI.

Citation: Gomez-Pinilla PJ, Farro G, Di Giovangiulio M, Stakenborg N, Némethova A, et al. (2014) Mast Cells Play No Role in the Pathogenesis of Postoperative
Ileus Induced by Intestinal Manipulation. PLoS ONE 9(1): e85304. doi:10.1371/journal.pone.0085304
Editor: Shree Ram Singh, National Cancer Institute, United States of America
Received September 3, 2013; Accepted November 25, 2013; Published January 9, 2014
Copyright: ß 2014 Gomez-Pinilla et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Supported by grants from Research Foundation - Flanders (FWO) (Odysseus program to GEB), by a FWO postdoctoral research fellowship (to GM and
PJG) by FWO PhD fellowship (to MDG), by DFG (SFB 983 project L to HRR) and by the ERC (grant no. 233074 to HRR). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: gianluca.matteoli@med.kuleuven.be

Introduction
Open abdominal surgery leads to impaired motility of the entire
gastrointestinal (GI) tract, a condition referred to as postoperative
ileus (POI) [1]. Depending on the type of surgery, POI may last
several days and, in up to 10% of patients, it might be prolonged
to over 2 weeks, with symptoms including nausea, vomiting,
intolerance to food and absence of defecation [2]. In addition to
significant patient morbidity, POI is associated with increased
hospital costs [3]. Therefore, any reduction in the occurrence or
duration of POI could lead to a significant reduction in
hospitalization and related costs.
POI is an immune-mediated condition characterized by a
localized inflammatory reaction in the muscularis externa evoked
by intestinal handling during surgery [1,4]. Macrophages residing
in the muscularis externa and mast cells have been proposed to be
the key players in this inflammatory cascade [1,4]. Pharmacolog-
ical or genetic (op/op mice) depletion of resident macrophages
indeed resulted in a decrease of inflammatory mediators and
diminished the recruitment of leucocytes into the muscularis
supporting a role for intestinal macrophages in the induction of
POI [5]. Activation of muscularis intestinal resident macrophages
subsequently leads to cytokine and chemokine release, followed by
an influx of leucocytes starting approximately 3–4 h after surgery
[6]. Finally, incoming leucocytes in conjunction with muscularis
macrophages (after surgery-related-IM) induce the synthesis of
prostaglandins and nitric oxide that directly impair smooth muscle
contractility and consequently lead to POI [7,8].

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In addition to activation of muscularis macrophages after
abdominal surgery, one of the earliest observations in rodents and
humans is the activation of peritoneal mast cells with the release of
their mediators into the peritoneal cavity [9,10]. The importance
of mast cells in the inflammatory cascade triggered by IM was also
suggested by experiments using mast cell stabilizers such as
ketotifen and doxantrazole. The treatment with the above
mentioned stabilizers reduced the inflammatory response and
the delay in gastrointestinal transit 24 h after abdominal surgery.
Moreover, mutant mice lacking mast cells (KitW/Wv and KitW-sh/W-
sh
) showed reduced intestinal infiltrate following IM while
reconstitution with wild type mast cells restored the phenotype
[9,11]. However, the role of mast cells in POI is not free of
criticism since the mediators measured (proteases and tryptases)
can be released even by other immune cells, while the mast cell
degranulation agonist (compound 48/80) and stabilizers (ketotifen
and doxantrazole) used are not specific for mast cells. In addition
to the specificity of the compounds tested, the use of mast cells
deficient mice based on Kit mutations is ambiguous, as the strains
used (KitW/Wv and KitW-sh/W-sh) have alterations in multiple cell
types of both immune and non-immune origin in addition to the
mast cell defect [12–17]. In particular, Kit is necessary for the
development of interstitial cells of Cajal (ICC), with both KitW/Wv
and KitW-sh/W-sh strains having severe alteration of the ICC
networks in the intestinal wall [15–17], and thus these mutations
may cause mast cell-independent defects in gut motility.
To avoid this experimental bias in the current study, we used a
genetic modified mouse strain with a targeted insertion of Cre-
recombinase into the Carboxypeptidase A3 (Cpa3) locus (Cpa3Cre/+
mice). This intervention leads to the specific mast cell ablation in
tissues by a genotoxic transformation related protein 53 (Trp53)-
dependent mechanism [18,19]. In contrast to Kit mutants,
Cpa3Cre/+ mutants have a selective mast cell depletion and apart
from a reduction in basophil numbers, other subpopulations of
immune cells are intact [18]. Therefore, this new transgenic mouse
model gives us the opportunity to specifically evaluate the role of
mast cells in POI.
Here we show that KitW-sh/W-sh mice have impaired gut motility
at baseline due to the alterations on ICCs distribution, making this
mouse strain unsuitable to study the role of mast cells in POI. By
contrast, the selective depletion of mast cells (and partially of
basophils) does not affect GI motility and does not prevent the
development of IM-induced muscular inflammation and POI.
Taken together, our data indicate that mast cells are not crucial in
the development of POI.
Materials and Methods
Animals
Wild type mice (C57BL/6JOlaHsd; WT) were purchased from
Harlan. KitW-sh/W-sh mice were obtained by homozygote mating of
mice originally purchased from The Jackson Laboratory [20].
Cpa3Cre/+ gene-targeted mice have been described previously
[18,21]. Mice were kept at the KU Leuven animal facility under
SPF conditions. All experimental procedures were approved by
the Animal Care and Animal Experiments Committee of the
Medical Faculty of the KU Leuven (Leuven, Belgium).
Surgical procedure to induce postoperative ileus
Mice were anesthetized by intraperitoneal injection (i.p.) of a
mixture of Ketamine (Ketalar 100 mg/kg; Pfizer) and Xylazine
(Rompun 10 mg/kg; Bayer). Anesthetized mice underwent a
laparotomy alone or a laparotomy followed by IM [9,22–24].
Surgery was performed using a sterile moist cotton applicator
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Mast Cells Are Not Involved in Postoperative Ileus
attached to a device enabling the application of a constant pressure
of 90 mN [25]. The small intestine was manipulated three times
from the caecum to the distal duodenum. During and after the
surgical procedure, mice were positioned on a heating pad (32uC)
until they recovered from anesthesia. No pharmacological
treatment was used to avoid influence on the outcome of the study.
Gastrointestinal transit measurements
To assess GI transit, 10 ml of a liquid non-absorbable fluorescein
isothiocyanate-labeled dextran (FITC-dextran, 70,000 Da; Invi-
trogen) was administered intragastrically 24 hours postoperatively
to fasted animals. Ninety minutes after oral gavage, animals were
sacrificed by CO2 overdose and the contents of stomach, small
bowel (divided into 10 segments of equal length), caecum, and
colon (divided in 3 segments of equal length) were collected and
the amount of FITC in each bowel segment was quantified using a
spectrofluorimeter (Ascent, Labsystem) at 488 nm. The distribu-
tion of the fluorescent dextran along the GI tract was determined
by calculating the geometric center (GC): S (percent of total
fluorescent signal in each segment x the segment number)/100 for
quantitative comparison among experimental groups [5].
Whole mount preparation and MPO staining
Twenty four hours after surgery, mice were sacrificed by CO2
overdose. The jejunum was quickly excised, and the mesenteric
attachment removed. Jejunum segments were cut open along the
mesentery border, fecal content was washed out in ice-cold
modified Krebs solution, and segments were fixed with 100%
ethanol for 10 minutes. Next, the mucosa and submucosa were
removed and the remaining full-thickness sheets of muscularis
externa were stained with Hanker Yates reagent (Sigma-Aldrich)
for 10 minutes [26]. Myeloperoxidase (MPO) positive cells were
visualized with a microscope (BX 41, Olympus) connected to a
camera (XM10, Olympus). The number of MPO-positive cells was
counted by an observer blind to the experimental conditions in 10
randomly chosen representative low-power magnification fields
(acquired with the 10X objective, 668.4 mm x 891.2 mm).
Staining and immunolabeling of mast cells, ICCs and
intestinal muscularis macrophages
Mesenteric windows were carefully preserved and pinned down
in a sylgard dish and subsequently fixed with 4% paraformalde-
hyde (Sigma-Aldrich) in PBS at 4uC for 10 minutes. To stain mast
cells, mesenteric windows were incubated with 0.1% of toluidine
blue (Sigma-Aldrich) for 1 hour and washed in PBS 3 times for 5
minutes.
Jejunum fragments were fixed with 4% paraformaldehyde and
frozen in optimal cutting temperature compound (OCT; Neg 50;
Thermo Scientific). Jejunum tissues were cut in 10-mm-thick
transversal sections. After blocking for 2 hours at room temper-
ature in 1% bovine serum albumin (BSA; Sigma-Aldrich) in PBS,
sections were incubated with the primary antibodies rat anti Kit
(clone 2B8; eBioscience) and rabbit anti Ano1 (Abcam) at a
concentration of 1:500 in 0.3% (vol/vol) Triton X-100 plus 1%
BSA in PBS overnight at 4uC. Subsequently, sections were
incubated with the appropriate secondary antibodies donkey anti-
rat conjugated with CY5 (Jackson ImmunoResearch) and donkey
anti-rabbit conjugated with CY3 (Jackson ImmunoResearch) at a
concentration of 1:1000 in 0.3% (vol/vol) Triton X-100 plus 1%
BSA in PBS for one hour at room temperature. Sections were then
counterstained with 49,6-diamidino-2-phenylindole dilactate
(DAPI; Invitrogen) to label nuclei.
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Immunolabeling of intestinal resident macrophages was per-
formed as follows. Jejunum muscularis externa whole mount
preparations from naı̈ve animals were subjected to two hours
incubation with 1% bovine serum albumin (BSA, Sigma-Aldrich,
St. Louis, MO) at room temperature (RT). After blocking, the
preparations were incubated overnight with the primary antibody
rat anti-F4/80 (1:500, Biolegend) in PBS containing 1% BSA and
0.3% Triton X-100. The next day, the tissues were incubated for
1 hour at room temperature with the secondary antibody donkey
anti-rat CY5 conjugated (1.1000, Jackson ImmunoResearch).
Immunolabeled tissues were examined with an Olympus BX4
epifluorescence microscope (Olympus). Contrast and brightness of
the pictures were adjusted using Image J software 1.46.
Quantification of mouse mast cell protease-1 in
peritoneal lavage fluid
Peritoneal lavage fluid was collected 30 minutes after IM by
injection of 1 ml of warm sterile saline solution and a gentle
massage of the peritoneum for 30 seconds. After that, peritoneal
lavage fluid was collected and centrifuged at 300 g for 5 minutes at
4uC. The pellet was discarded and supernatant stored at 280uC
until use. Peritoneal levels of mouse mast cell protease-1 (mMCP-
1) as a measure of mast cell degranulation were measured by using
a commercially available ELISA kit (eBioscience) following
manufacturer’s instructions. mMCP-1 levels were normalized to
the protein concentration in the peritoneal lavage fluid.
RNA extraction and inflammatory gene expression
Total RNA was extracted from the muscularis externa of the
jejunum 24 hours after surgery. To this extent, tissue was
homogenized by the TissueLyser II homogenizer (Qiagen). RNA
extraction was performed using RNeasy Mini Kit (Qiagen)
following the manufacturer’s instructions. Total RNA was
transcribed into complementary DNA (cDNA) by qScript cDNA
SuperMix (Quanta Biosciences) according to the manufacturer’s
instructions. Quantitative real-time transcription polymerase chain
reactions (RT-PCR) were performed with the LightCycler 480
SYBR Green I Master (Roche) on the Light Cycler 480, (Roche).
Results were quantified using the 2-DDCT method [27]. The
expression levels of the genes of interest were normalized to the
expression levels of the reference gene rpl32. PCR experiments
were performed in triplicate. Primer sequences used are listed in
Table S1.
Cell isolation from the intestinal muscularis for flow
cytometry
Twenty-four hours after the surgery, muscularis externa from
the small intestine was isolated and enzymatically digested in
MEMa medium (Lonza) containing 100 mg/ml of Penicillin,
100 mg/ml of Streptomycin, 50 mM b-mercaptoethanol, 5% FCS,
5 mg/ml protease type I (Sigma-Aldrich), 20 mg/ml collagenase
type II (Sigma-Aldrich) and 5 U/ml DNase I for 15 min at 37uC.
Cell suspensions were pre-incubated with an anti-FcR antibody
(clone 24G2; BD Biosciences) and then stained with the following
antibodies: CD45-APC-eFluor780 (30.F11, eBioscience), CD11b-
PE-Cy7 (M1/70, BD Biosciences), CD64-Alexa Fluor647 (X54-5/
7.1, BD Biosciences), Ly6G-PercPCy5.5 (IA8, BD Biosciences) and
Ly6C-PE (AL-21, BD Biosciences). Stained samples were analyzed
on a BD FACSCantoTM flow cytometer (BD Biosciences), and
data were processed using FlowJo software (version 10.0.6, Tree
Star). Cell numbers were calculated from flow cytometry
frequencies using hemocytometer counts of trypan blue–excluding
cells.
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Mast Cells Are Not Involved in Postoperative Ileus
Cromolyn administration
Mice received cromolyn (Disodium cromoglycate, NalcromH,
Italchimici) by intraperitoneal injection of 30 mg/kg (in sterile
saline solution) every 12 hours. The animals received cromolyn at
three time points; 13 h and 1 h before IM and 11 h after IM
(n = 10). Another group of mice (n = 10) received 200 ml of sterile
solution (vehicle) at the same time points than cromolyn treated
group. The researcher performing the surgeries was blinded for
the type of pharmacological treatment.
Statistical analysis
To compare multiple groups, one-way analysis of variance (one-
way ANOVA) followed by Bonferroni post-hoc test was per-
formed. Probability level of p,0.05 was considered statistically
significant. Results are shown as mean 6 standard error of the
mean (SEM). Graph Pad Prism V.5.01 software was used to
perform statistical analysis and generate graphs.
Results
Intestinal manipulation induces peritoneal mast cell
degranulation
To define if peritoneal mast cell degranulation was induced
during IM, we performed toluidine staining and quantified mouse
mast cell protease-1 (mMCP-1) release in the peritoneal cavity of
WT and KitW-sh/W-sh mice.
Toluidine blue staining showed cells with typical dark-blue or
purple cytoplasmic granules resembling mast cells in the mesen-
teric window of WT mice (Figure 1A). As expected, no mast cells
were found in the mesenteric windows from KitW-sh/W-sh mice
(Figure 1A). IM induced typical signs of degranulation in WT
mice, as visualized in Figure 1A by the appearance of dark-blue
(toluidine blue-positive) structures released from and in the
surrounding of a mast cell in the mesenteric window. In line, in
the peritoneal lavage fluid of WT mice significant amount of
mMCP-1 was detected already 30 minutes after IM (Lap;
0.03660.0011 vs Lap +IM; 0.99060.483 ng/ml; Figure 1B). As
control, IM in the mast cell-deficient KitW-sh/W-sh mutant mice did
not lead to increase in peritoneal levels of mMCP-1 (Lap;
0.04460.0013 vs Lap + IM; 0.03960.009 ng/ml; Figure 1B).
As previously reported, IM in WT mice resulted in a significant
delay in gastrointestinal transit (as measured by a reduction in the
geometric center values, GC) compared to laparotomy (Figure 1C).
In line with our previous observations IM led to recruitment of
MPO-positive cells to the muscularis externa (Figure 1D). To
define the role of mast cells in the pathogenesis of POI, IM was
performed also in KitW-sh/W-sh mutant mice. As shown in Figure 1C,
gut transit was already significantly delayed in KitW-sh/W-sh mutants
undergoing laparotomy compared to control WT mice and IM did
not worsen gastrointestinal transit in KitW-sh/W-sh mice when
compared to their laparotomy controls.
Interestingly, IM in KitW-sh/W-sh mice resulted however in
recruitment of MPO-positive cells to the muscularis externa with
the same extent as in WT mice (WT; 143618 number of cells per
field vs KitW-sh/W-sh 95620 number of cells per field, ns, Figure 1D).
Since IM induced recruitment of MPO-positive cells even in the
absence of mast cells (KitW-sh/W-sh) we analyzed the inflammatory
response in the muscularis externa by assessing mRNA cytokine
expression and the recruitment of immune cells. In line with the
number of MPO-positive cells IM significantly increased cytokine
mRNA expression (Il6, Il1a, Il1b, Tnfa, Cxcl1 and Ccl2; Figure 2)
in the muscularis externa of KitW-sh/W-sh mice when compared to
laparotomy mice.
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 Mast Cells Are Not Involved in Postoperative Ileus

Figure 1. Intestinal manipulation in KitW-sh/W-sh mice induces intestinal inflammation in the absence of mast cells. WT and KitW-sh/W-sh
mice were subjected to laparotomy alone (Lap) or to laparotomy plus intestinal manipulation (Lap + IM). (A) Mesenteric windows from WT and KitW-sh/
W-sh
mutant mice were collected 30 min after surgeries and stained with 0.1% toluidine blue. Scale bar 50 mm. (B) 30 minutes after surgery the levels
of mouse mast cell protease-1 (mMCP-1) was determined by ELISA in the peritoneal lavage fluid of WT and KitW-sh/W-sh. (C) Geometric center (GC)
values representing the dextran distribution through the GI tract 24 hours after surgery. (D) Infiltration of MPO-positive cells in the muscularis externa
24 hours after surgery. Data expressed as mean 6 SEM. * P,0.01 (one-way ANOVA followed by Bonferroni post-hoc test). Dots represent individual
mice.
doi:10.1371/journal.pone.0085304.g001

Intestinal manipulation-induced recruitment of immune cells to
the muscularis is a typical event in POI. Therefore, we determined
by flow cytometry the recruitment of immune cells in WT and
KitW-sh/W-sh mutant mice after laparotomy or laparotomy plus
intestinal manipulation. As shown in Figure 3, IM induced a
significant increase in CD45-positive immune cells, monocytes and
neutrophils in the small intestine muscularis both in WT and
KitW-sh/W-sh mutant mice. Interestingly, no difference in the
percentage and in the absolute number of monocytes and
neutrophils were detected between WT and KitW-sh/W-sh mutant
mice (Figure 3C–D).
Taking into account that muscularis externa resident macro-
phages are suggested to be key players in the development of
postoperative ileus in rodents and humans we performed F4/80
immunolabeling in jejunum whole mount muscularis preparations
from WT and KitW-sh/W-sh mutant mice. As shown in Figure S1
distribution and number of F4/80-positive resident macrophages
are comparable in both WT and KitW-sh/W-sh mutant mice.
KitW-sh/W-sh mice lack ICCs and have altered
gastrointestinal transit
Taking into account that Kit signaling is essential for the
development of ICCs and that gut motility is impaired in
KitW-sh/W-sh mutant mice we performed immunolabelling of the
ICC networks in WT and KitW-sh/W-sh mice. As previously reported
in the jejunum of WT mice, anti-Kit antibody labeled ICCs
located at the level of the deep muscular and myenteric plexus
regions of the muscularis externa (Figure 4A), as well as mast cells
(white arrows Figure 4A, panel right). However, no Kit positive
cells, mast cells or ICCs, were found in the muscularis externa
from KitW-sh/W-sh mutant mice (Figure 4B). To search for the
presence of ICCs independently of Kit staining in KitW-sh/W-sh
mice, we used the recently described ICC marker Anoctamin-1
(Ano1) [28]. In the jejunum of WT mice, Ano1 co-stained with Kit
specifically the networks of ICCs located at the deep muscular and
at the myenteric plexus regions (Figure 4A). By contrast, in the
small bowel of KitW-sh/W-sh mutant mice, Ano1 immunoreactivity
was found exclusively at the level of the deep muscular plexus
region (Figure 4B), where only few cells with typical ICC
morphology were found (Figure 4B). Our results confirm a
malformation of the ICC networks in the KitW-sh/W-sh mutant mice
(Figure 4A and B). This alteration in the number and distribution
of the ICCs in the KitW-sh/W-sh mutant mice was associated with
significant gut dysmotility found in both naı̈ve mice and mice
subjected to laparotomy (Figure 4C and D). Therefore, we
conclude that KitW-sh/W-sh is not an adequate mouse model to study
the role of mast cells in POI.
Mast cell-deficient Cpa3Cre/+ mice had normal ICC
network and regular gastrointestinal transit
Considering that naı̈ve untreated KitW-sh/W-sh mice have already
severe alteration of the gut motility we decided to utilize in our

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 Mast Cells Are Not Involved in Postoperative Ileus

Figure 2. Intestinal manipulation in KitW-sh/W-sh mice induces cytokines expression in the absence of mast cells. WT and KitW-sh/W-sh mice
were subjected to laparotomy alone (Lap) or to laparotomy plus IM (Lap + IM). Twenty four hours after surgery muscularis externa was collected and
cytokines mRNA expression assessed by qPCR. Il6 (A), Il1a (B), Il1b (C), Tnfa (D), Cxcl1 (E) and Ccl2 (F) mRNA expression was evaluated in the jejunum
muscularis externa after 24 h. Data expressed as mean 6 SEM. * P,0.05, ** P,0.01 or *** P,0.001 (one-way ANOVA followed by Bonferroni post-hoc
test). Dots represent individual mice.
doi:10.1371/journal.pone.0085304.g002

study a newly described mast cell–deficient mouse strain Cpa3Cre/+
with non-perturbed Kit functions [18]. Using toluidine staining
and Kit immunolabelling we observed a lack of mast cells both in
the peritoneal cavity (Figure 5A) and in the intestinal mucosa
(Figure 5B). Interestingly, and contrast to previously described Kit
mast cell-deficient mice, Kit labeling of the muscularis externa
revealed the presence of a normal network of ICCs in the deep
muscular plexus and in the myenteric regions in Cpa3Cre/+
(Figure 5D), with no relievable differences compared to littermate
control Cpa3+/+ mice (Figure 5C). To assess whether the presence
of normal ICCs in the absence of mast cells is associated with
normal gut motility, we performed gastrointestinal transit analyses
in Cpa3Cre/+ mice without surgery or only after laparotomy. As
depicted in Figure 5E and F, Cpa3Cre/+ mice have normal GI

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 Mast Cells Are Not Involved in Postoperative Ileus

Figure 3. Intestinal manipulation in KitW-sh/W-sh mice induces recruitment of immune cells in the muscularis externa in the absence of
mast cells. WT and KitW-sh/W-sh mice were subjected to laparotomy alone (Lap) or to laparotomy plus intestinal manipulation (Lap + IM) and immune
cells recruitment in the muscle layer of the small intestine was assessed by flow cytometry. Typical dots plot showing different population of CD45-
positive cells in WT and KitW-sh/W-sh mice after (A) laparotomy or (B) laparotomy plus intestinal manipulation. Absolute number of CD45 positive
immune cells (C), monocytes (D) and neutrophils (E) were calculated from flow cytometry frequencies using viable cell counts. Data expressed as
mean 6 SEM. ** P,0.01 or *** P,0.001 (one-way ANOVA followed by Bonferroni post-hoc test). Dots represent individual mice.
doi:10.1371/journal.pone.0085304.g003

transit with no significant difference when compared to Cpa3+/+
littermate or to WT mice. In addition, as shown in Figure S1 the
distribution and number of F4/80-positive muscularis externa
resident macrophages are comparable in both Cpa3Cre/+ and
littermate control mice.
Mast cell-deficient Cpa3Cre/+ mice develop postoperative
ileus and surgery-induced muscularis externa
inflammation
To define if the presence of mast cells has an influence on the
development of POI we performed intestinal manipulation in mast
cell-deficient Cpa3Cre/+ mice and in Cpa3+/+ littermate controls.
Despite the absence of mesenteric as well as intestinal mast cells
(Figure 5A and B), IM in Cpa3Cre/+ mice induced a delay in GI
transit as shown by a reduction in GC value (Cpa3Cre/+ lap; GC:
10.260.2 vs Cpa3Cre/+ IM, GC: 4.160.3). The extent of this delay
was indistinguishable to littermate control Cpa3+/+ mice (Cpa3+/+
lap; GC: 10.260.3 vs Cpa3+/+ IM, GC: 4.260.6) (Figure 6A).
Next, peritoneal levels of mMCP-1 were assessed in Cpa3Cre/+ and
Cpa3+/+ mice 30 minutes after IM. Consistent with their mast cell
deficiency, IM did not increase peritoneal mMCP-1 in Cpa3Cre/+
mice (Lap; 0.07460.0023 vs Lap + IM; 0.03660.008 ng/ml;
Figure 6B) while Cpa3+/+ had a significant increase of this protein
in IM versus laparotomy (Lap; 0.07660.0015 vs Lap + IM;
1.25160.273 ng/ml; Figure 6B). Intestinal manipulation in mast
cell-deficient Cpa3Cre/+ mice also led to inflammatory response in
the muscularis externa based on influx of MPO-positive cells
(Cpa3Cre/+; 94614 number of cells per field; Figure 6C) with no
significant difference to littermate controls (Cpa3+/+; 113611
number of cells per field; Figure 6C). A normal inflammatory
response to IM in the absence of mast cells was also evident by the
fact that IM-induced mRNA levels for a range of inflammatory
cytokines (Il6, Il1a, Il1b, Tnfa, Cxcl1 and Ccl2) in the muscularis
externa were comparable in Cpa3Cre/+ mice and littermate controls
(Figure 7).
As previously reported intestinal manipulation leads to the
recruitment of neutrophils and monocytes in the muscularis
externa. To determine if the lack of mast cells would affect this
process, we assessed by flow cytometry the immune cells
infiltrating the small bowel muscularis of Cpa3Cre/+ and Cpa3+/+
mice 24 hours after laparotomy or laparotomy plus intestinal
manipulation. As evident in Figure 8, no difference in the

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 Mast Cells Are Not Involved in Postoperative Ileus

Figure 4. Deficient ICC network and intestinal dysmotility in KitW-sh/W-sh mice. ICC network in the intestinal wall and GI transit were assessed
in WT and KitW-sh/W-sh mice. Jejunum sections from naı̈ve WT mice (A) or KitW-sh/W-sh mice (B) were immunolabeled with anti-Kit (red) and anti-Ano1
(green) antibodies. Sections were counterstained with DAPI (blue) to identify nuclei. White arrows are pointing Kit-positive and Ano1-negative mast
cells in the jejunum from a WT mouse. DMP, deep muscular plexus; MYP, myenteric plexus; CM, circular muscle layer and LM, longitudinal muscle
layer. Scale bar 50 mm. Ninety min after oral gavage with dextran-FITC naı̈ve (C) or animals subjected to laparotomy (D) WT and KitW-sh/W-sh were
sacrificed and dextran-FITC distribution through the GI tract was determined as indicative of GI transit. Data expressed as mean. *** P,0.001 (two-
way ANOVA).
doi:10.1371/journal.pone.0085304.g004

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 Mast Cells Are Not Involved in Postoperative Ileus

Figure 5. Cpa3Cre/+ mice lack mesenteric and mucosal mast cells but have normal ICC network and gut motility. Naı̈ve Cpa3Cre/+ and
littermates control Cpa3+/+ mice were used to analyze intestinal mast cells, ICCs and GI transit. (A) Mesenteric windows from Cpa3+/+ and Cpa3Cre/+
mice were stained with 0.1% toluidine blue. Scale bar 50 mm. (B) Jejunum mucosa sections from naı̈ve Cpa3+/+ and Cpa3Cre/+ mice were
immunolabeled with Kit antibody (red) and counterstained with DAPI (blue). Scale bar 25 mm. White arrows are pointing to mast cells in Cpa3+/+ mice.
To reveal ICCs, jejunum sections from Cpa3+/+ (C) and Cpa3Cre/+ (D) mice were immunolabeled with Kit (red) and counterstained with DAPI (blue).
DMP, deep muscular plexus; MYP, myenteric plexus; CM, circular muscle layer and LM, longitudinal muscle layer. Scale bar 50 mm. GI transit was
evaluated in naı̈ve (E) or animal subjected to laparotomy (F) WT, Cpa3+/+ and Cpa3Cre/+ mice by assessing dextran-FITC distribution through the GI
tract during 90 min after oral gavage. Data are expressed as means. No significant differences were found between the groups of animals (two-way
ANOVA).
doi:10.1371/journal.pone.0085304.g005

percentage and in the absolute number of CD45-positive immune
cells, monocytes and neutrophils were found between Cpa3Cre/+
and littermate control Cpa3+/+ mice subjected to laparotomy or
laparotomy plus intestinal manipulation.
Cromolyn treatment inhibits mast cell degranulation but
does not prevent POI
The use of mast cell stabilizers is an additional strategy to
address the roles of mast cells under pathological conditions. Thus,
we tested the ability of the a typical mast cell stabilizer, cromolyn,
to influence the response to IM [29]. Cromolyn significantly

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 Mast Cells Are Not Involved in Postoperative Ileus

Figure 6. Intestinal manipulation induces postoperative ileus and recruitment of MPO-positive cells in the muscularis externa
independently of mast cells. Cpa3Cre/+ and littermates control Cpa3+/+ mice were subjected to laparotomy alone (lap) or to laparotomy plus IM
(lap + IM). GI transit was evaluated 24 h after surgery by assessing dextran-FITC distribution through the gastrointestinal tract 90 min after oral
gavage. (A) Graph represents GC values. (B) Peritoneal levels of mMCP-1 were determined by ELISA in Cpa3+/+ and Cpa3Cre/+ mice. (C) Representative
images of MPO-positive cells in the muscularis externa 24 h after surgery in Cpa3Cre/+ and littermates control Cpa3+/+ mice. (D) Histogram represents
numbers of MPO-positive cells in the muscularis externa 24 h after surgery in Cpa3Cre/+ and littermates control Cpa3+/+. Data expressed as mean 6
SEM. * P,0.01 (one-way ANOVA followed by Bonferroni post-hoc test). Dots represent individual mice.
doi:10.1371/journal.pone.0085304.g006

inhibited the release of mMCP-1 in the peritoneal cavity evoked
by IM (Figure S2A). Nevertheless, mice treated with cromolyn still
developed delayed GI transit following IM, similar to the vehicle
treated mice (Figure S2B).
Discussion
Recent evidence shows that postoperative ileus is mediated by
infiltration of leukocytes in the intestinal muscle layer in response
to surgical handling of the gut [8–10]. Activation of resident
macrophages and mast cells has been proposed to be involved in
this inflammatory response. However, we here demonstrate, using
the mast cell-deficient Cpa3Cre/+ mouse strain, that mast cells do
not have a crucial role in the pathogenesis of POI. In the current
experiments we used a new mouse strain devoid of both mucosal
and connective tissue subtypes of mast cells [18]. In this mouse
strain, Cre recombinase is driven by the Cpa3 locus which is
expressed in mast cells from their progenitor stage onwards.
Cpa3Cre/+ mice have been used previously to re-address the
proposed roles of mast cells in autoimmunity, refuting an
involvement of mast cells in the tested models of antibody and T
cell-mediated autoimmunity [18].
Considering that Kit signaling is crucial for the development of
normal ICC networks and intestinal motility, an important read-
out in our POI model, it was critical to use mice with normal
ICCs. Indeed, Cpa3Cre/+ mice, in contrast to previously used mast
cell-deficient strains, have intact Kit signaling. Consequently, we
found normal ICC networks and regular intestinal transit time in
Cpa3Cre/+ mice (Figure 5). Moreover, the immune system is not
compromised (with the exception of a lower number of basophils)
in Cpa3Cre/+ mice, whereas other Kit mutant mast cell-deficient
strains have deficiencies in several immune cell subtypes or their
functions. Clearly, although no mast cells could be demonstrated
based on histology and mast cell mediator release following
surgery, Cpa3Cre/+ developed IM-induced intestinal inflammation
and delay of gastrointestinal transit, the two hallmark features of
POI, to the same extent as Cpa3+/+ littermate mice (Figure 6). The
fact that Cpa3Cre/+ mice developed full POI strongly argues against
mast cells as crucial player in the development of POI.
In contrast to our new data, a role for mast cells in POI had
been invoked based on previous findings. First, mast cell products
such as tryptase were released in the peritoneal cavity after
intestinal manipulation both in rodents and human [9,10].
However, in addition to mast cells other immune cells such as
basophils and neutrophils may also be a source of tryptase [30].
Second, the mast cell secretagogue compound 48/80 (C48/80)
was used to provoke mast cell activation leading to muscularis
inflammation, as indicated by an increase in MPO-positive cell
infiltration. However, an effect on gut motility was not assessed
under these conditions [9]. Moreover, C48/80 has other non-
specific effects amongst which inducing oxidative stress [31],vaso-

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 Mast Cells Are Not Involved in Postoperative Ileus

Figure 7. Intestinal manipulation induces muscularis externa inflammation independently of mast cells. Cpa3Cre/+ and littermate
control Cpa3+/+ mice were subjected to laparotomy alone (Lap) or to laparotomy plus IM (Lap + IM). Twenty four hours after surgery muscularis
externa was collected and cytokines mRNA expression assessed by qPCR. Il6 (A), Il1a (B), Il1b (C), Tnfa (D), Cxcl1 (E) and Ccl2 (F) mRNA expression was
evaluated in the jejunum muscularis externa after 24 h. Data expressed as mean 6 SEM. * P,0.01 (one-way ANOVA followed by Bonferroni post-hoc
test). Dots represent individual mice.
doi:10.1371/journal.pone.0085304.g007

dilatation [32], and it acts on other immune and non-immune cells
including basophils [33], neurons [34,35] and fibroblasts [36]. In
line with these findings, the interpretation of the data acquired
using the mast cell stabilizers such as ketotifen or doxantrazole
should be used with caution, considering their broad anti-
inflammatory effect mainly independent of mast cells [37–40].
In previous studies reconstitution experiments with wild type mast
cells were used to restore the phenotype which suggested a role for
mast cells in POI. Accumulating evidences, however, question this
approach. It is now clear that although bone marrow-derived mast

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 Mast Cells Are Not Involved in Postoperative Ileus

Figure 8. Intestinal manipulation induces recruitment of immune cells in the muscularis externa in the absence of mast cells.
Cpa3Cre/+ and littermate control Cpa3+/+ mice were subjected to laparotomy alone (Lap) or to laparotomy plus intestinal manipulation (Lap + IM) and
immune cells recruitment in the muscle layer of the small intestine was assessed by flow cytometry. Typical dots plot showing different population of
CD45-positive cells in Cpa3Cre/+ and littermate control Cpa3+/+ mice after (A) laparotomy or (B) laparotomy plus intestinal manipulation. Absolute
number of CD45 positive immune cells (C), monocytes (D) and neutrophils (E) were calculated from flow cytometry frequencies using viable cell
counts. Data expressed as mean 6 SEM. *** P,0.001 (one-way ANOVA followed by Bonferroni post-hoc test). Dots represent individual mice.
doi:10.1371/journal.pone.0085304.g008

cells (BMMCs) can adopt the phenotype of normal tissue mast cells
after in vivo transfer, numbers, distribution, and functional
responses of reconstituted mast cells may not always be
physiological [19]. Indeed, several reports using BMMC reconsti-
tution showed a reversal of the phenotype in Kit mutant mice but
this has not been confirmed when tested independently in mast
cell-deficient mice wild type for Kit. Hence, mast cell reconstitu-
tion of Kit mutants does not necessarily reflect mast cell functions
in the presence of intact Kit signaling, and may lead to
misinterpretation of experimental data and incorrect conclusions
[19]. Collectively, none of these previous experiments directly and
conclusively proved mast cells to be involved in intestinal
pathology.
In addition to these pharmacological studies, the role of mast
cells in POI has been traditionally investigated using mice that are
mast cell-deficient due to impaired Kit signaling, such as KitW/Wv
and KitW-sh/W-sh mice. However, these mouse strains have
pleiotropic phenotypes affecting multiple cell types which comprise
normal tissue functions inside and outside of the immune system,
as reviewed in [19]. For example, KitW/Wv mice suffer from anemia
and neutropenia, and lack subsets of intra-epithelial lymphocytes.
A strain more recently used in mast cell research, KitW-sh/W-sh mice,
displays a milder spectrum of abnormalities, but is also affected by
immunological abnormalities such as splenomegaly, accumulation
of granulocytic myeloid-derived suppressor cells (G-MDSC) [12],
expanded myeloid and megakaryocyte populations, neutrophilia
and thrombocytosis [13,14]. These defects in KitW-sh/W-sh mice
may have contributed to the reported lower inflammatory
response, and the reduced number of MPO-positive cells in the
muscularis externa after intestinal manipulation [9]. However,
here we provide evidence that in KitW-sh/W-shintestinal manipula-
tion induced muscularis externa inflammation (increase in
cytokine expression and recruitment of monocytes and neutro-
phils) even in the absence of mast cells (Figure 1 to 3).
Given that ICC networks in the intestinal wall fail to develop
normally in Kit mutant mice [15–17], it is not surprising to
observe a significant delay in the gastrointestinal transit in naı̈ve
KitW-sh/W-sh mice (Figure 4). As gastrointestinal transit is one of the

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major parameters of POI, the ICCs defect should exclude Kit-
based mouse models for studying the pathogenesis of ileus. In
contrast to Kit mutants, Cpa3Cre/+ mice have intact ICC network
and normal intestinal motility (Figure 5) and their immune system
is not compromised [18]. Notably, mast cell-deficient Cpa3Cre/+
mice developed IM-induced intestinal inflammation and delay of
gastrointestinal transit, the two hallmarks of POI, to the same
extent as their mast cell-proficient littermates, indicating that mast
cells are not involved in POI (Figure 6 to 8). In support of this
conclusion, cromolyn treatment (30 mg/kg; a dose which has been
proved to stabilize mast cells in vivo [29]) was ineffective in
preventing POI (Figure S2).
In conclusion, our study demonstrates that mast cells, at least in
mice, do not play a crucial role in the development of POI, and
provide a further example of experimental discrepancies compar-
ing mast cell- and Kit double-deficient mutants versus mast cell-
deficient mice without defects in Kit signaling as discussed in [19].
These findings should be definitively taken into account when
designing new therapeutic strategies to shorten POI.
Supporting Information
Figure S1 Cpa3Cre/+ mice lack mesenteric and muco-
sal mast cells but have normal ICC network and gut
motility. Naı̈ve WT, KitW-sh/W-sh or Cpa3Cre/+ and
littermates control Cpa3+/+ mice were used to analyze the
network of resident muscularis externa macrophages. Muscularis
externa isolated from the jejunum of WT or KitW-sh/W-sh mice
(A) or Cpa3Cre/+ and littermates control Cpa3+/+ mice (B) was
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Mast Cells Are Not Involved in Postoperative Ileus
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(PDF)
Figure S2 Cromolyn treatment inhibits mast cell de-
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hoc test). Dots represent individual mice.
(PDF)
Table S1 Primers list.
(PDF)
Acknowledgments
We would like to thank R. De Keyser and I. Croux for their excellent
technical assistance.
Author Contributions
Conceived and designed the experiments: PJG-P H-RR GEB GM.
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the data: PJG-P GM. Contributed reagents/materials/analysis tools: H-
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